Lecithin-cholesterol acyltransferase (LCAT) is a glycoprotein that is responsible for the synthesis of most cholesteryl esters (CE) in plasma. We have found that dietary long chain polyunsaturated fatty acids (PUFA) decrease plasma LCAT reactivity, but do not alter enzyme mass, and decrease the size of plasma HDL subfractions. Based on our preliminary studies we hypothesize that long chain PUFA decrease CE formation by LCAT by: 1) decreasing the binding of lCAT to the interface of HDL particles and/or 2) by decreasing the turnover of monomeric phosphatidylcholine (PC) substrate at the active site of the enzyme. The late=ter effect appears related to the molecular size of the PC molecules, since we have observed an inverse relationship between PC molecular area on monolayers and LCAT phospholipase A2 activity. To test our hypotheses we will accomplish 2 specific aims. In the first specific aim, we will determine the regions of the LCAT protein that are important in determining the fatty acyl substrate specificity of LCAT. We will take advantage of the difference in substrate specificity between rodent and primate LCAT. Rodent LCAT preferentially uses long chain PUFA to generate CE whereas primate LCAT uses primarily 18:1 and 18:2. We hypothesize that this difference in specificity is due to a larger active site pocket in rat LCAT that can accommodate larger PC substrate molecules compared to the primate LCAT. Site-directed mutagenesis will be used to systematically change the primary sequence of the human cDNA to that of the rat cDNA at regions of sequence divergence between the primate and rodent protein. Wild type human and rat and mutant LCAT cDNA constructs will be transfected into COS cells and LCAT protein secreted into the media will be tested for phospholipase A2 activity and CE synthesis using recombinant HDL (rHDL) with PC molecules containing different fatty acyl species in the sn-2 position (18:1, 18:2, 20:5 n-3, 22:6 n-3); a water soluble substrate will be used to control for any global effects of the mutagenesis on LCAT reactivity. Mutant constructs of LCAT that exhibit increased reactivity to long chain PUFA will be overexpressed in a baculovirus system. In specific aim 2 wild type human and rat and mutant LCAT proteins, derived from a baculovirus expression system, will be tested for differences in interfacial binding (affinity and capacity) to monolayers and vesicles of PC, rHDL, and plasma and liver perfusate HDL from nonhuman primates consuming 4 types of dietary fat. The monolayer, vesicle and rHDL studies will be performed with PC species containing different fatty acids in the sn-2 position as well as plasma PC from each of th diet groups. These studies should help define the mechanism(s) by which PC fatty acyl composition modulates LCAT reactivity and ultimately plasma HDL subfraction distribution. The data from these studies will be important in determining the influence of dietary fat modification on the role of LCAT in maturation of nascent HDL particles, in the redistribution of plasma HDL subfractions, and in the reverse cholesterol pathway.